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Mycobiology 40(1) : 35-41 (2012) http://dx.doi.org/10.5941/MYCO.2012.40.1.035© The Korean Society of Mycology pISSN 1229-8093

eISSN 2092-9323

RESEARCH ARTICLE

Study of Sugarcane Pieces as Yeast Supports for Ethanol Production fromSugarcane Juice and Molasses Using Newly Isolated Yeast from Toddy Sap

Neerupudi Kishore Babu1*, Botcha Satyanarayana1, Kesavapillai Balakrishnan2, Tamanam Raghava Rao1 and

Gudapaty Seshagiri Rao1

1Department of Biochemistry, Andhra University, Visakhapatnam, Andhra Pradesh 530 003, India2Biotechnology Group, BIT, Sathyamangalam, Tamil Nadu 638 401, India

(Received November 1, 2011. Revised November 29, 2011. Accepted January 18, 2012)

A repeated batch fermentation system was used to produce ethanol using Saccharomyces cerevisiae strain (NCIM 3640)immobilized on sugarcane (Saccharum officinarum L.) pieces. For comparison free cells were also used to produce ethanolby repeated batch fermentation. Scanning electron microscopy evidently showed that cell immobilization resulted in firmadsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure,and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supportedbiocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sug-arcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and feweryeast cells leaked into fermentation broth. Ethanol concentrations (about 72.65~76.28 g/L in an average value) and ethanolproductivities (about 2.27~2.36 g/L/hr in an average value) were high and stable, and residual sugar concentrations were lowin all fermentations (0.9~3.25 g/L) with conversions ranging from 98.03~99.43%, showing efficiency 91.57~95.43 and oper-ational stability of biocatalyst for ethanol fermentation. The results of the work pertaining to the use of sugarcane as immo-bilized yeast support could be promising for industrial fermentations.

KEYWORDS : Ethanol production, Immobilization, Repeated batch fermentation, Saccharomyces cerevisiae, Sugarcane pieces

Introduction

Because of the increasing demand for ethanol, there is aneed to search for high-yielding strains and less expensivetechnology for production of ethanol, so that it can bemade available at a cheaper rate [1, 2]. One such processis yeast cell immobilization because of its technical andeconomical advantages compared to free cell system,which facilitates faster fermentation rates by providinghigher cell densities per unit fermentation volume. The insitu removal of cells further reduces the cost of recovery[3]. It also helps in protecting cells from the toxic effectsof low pH, temperature, osmotic, inhibitors, etc. andthereby increasing ethanol yield and reducing the costsrequired for inoculation development [4]. Further, thereduction of costs, in bioprocesses involving immobilizedcells systems are related to aspects such as the cost ofraw materials, the use of cheap, abundant and stableimmobilization supports, the high cell concentrations inthe bioreactors, the simplicity and low cost of theimmobilization techniques and the stability of the

immobilized biocatalyst in operating conditions [5].Various immobilization supports for variety of products

have been reported such as alginates [4, 6], apple pieces[7-10], orange peel [11], gluten pellets [12], sugar canepieces [13-15], guava piece and watermelon pieces [16, 17]and delignified cellulosic residues [18, 19]. Generally, thelignocellulose materials were delignified prior to applicationfor the immobilization supports. Natural cellulose materialscontained a large number of hydrophilic groups in the formof positive charge, ready for the absorption of negativecharged cells. Through the delignification treatment, thelignin was removed partially and leading to exposing morehydrophilic groups, which increased the absorption [20].The delignification treatment with sodium hydroxide solutionincreased the possibility cells going through the vesicles ofcut pieces and so accommodated there for immobilization[18].

Immobilization techniques used in general can begrouped into four categories according to Tanaka andKawamoto [21]. The first one includes those methodswhich involve the binding of the biocatalyst to a water-

*Corresponding author <E-mail : neerupudi@gmail.com>

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium,provided the original work is properly cited.

36 Babu et al.

insoluble support, by using ionic or covalent chemicallinks, bio specific coupling, or junctions due to adsorptionphenomena. Natural polymers such as polysaccharides(cellulose, dextran, and agarose derivatives), proteins(gelatine and albumin), synthetic polymers (polystyrenederivatives and polyurethane) and inorganic material (sand,clay, ceramics, and magnetite) are commonly used for thispurpose. A second category includes those methods usingmultifunctional compounds as glutaraldehyde, toluene orhexamethylene diisocyanate, to form Schiff’s bases withfunctional groups in biocatalysts, thus producing water-insoluble agglomerate of catalysts [14].

The third category is constituted by those methodsinvolving the trapping of the biocatalyst into a networkformed by one or several polymers (polyacrylamide, alginate,carraginate, or synthetic resins), or those involving theembedding in membranes, encapsulating them insidemicrocapsules composed of synthetic polymers. Acombination of the three former methods constitutes thelast category. As we known, the adhesion of hydrophiliccells such as Saccharomyces cerevisiae is essentiallydependent upon electrostatic interactions between thesupport and the normally negatively charged cell surface.

A strain of S. cerevisiae was isolated from toddy sap,which was found to produce significant amounts ofethanol, was deposited at National Collection of IndustrialMicroorganisms (NCIM), Pune, India and designated asNCIM 3640. In search of a biocatalyst that is suitable forindustrial use, is cheap and abundant, and could be useda number of times, we thought of using sugarcane.Sugarcane is one of the abundant crops available in India.Incidentally, India is the second largest producer ofsugarcane in the world (315.5 MT in 2008; FAO, USA).Its production is high during summer (April to July)because of its tropical nature and available throughout theyear. Sugarcane is a natural, abundant, cheap material andit is a suitable substrate for cell growth, therefore, wedecided to use sugarcane pieces as support for yeastimmobilization and then utilize it for alcohol productionfrom sugarcane juice and molasses by repeated batchfermentation and do not need special treatment before use.The aim of this study was to immobilize S. cerevisiaeNCIM 3640 on sugarcane pieces and evaluated theefficiency and suitability of the immobilized S.cereviseaefor ethanol production.

Materials and Methods

Materials, media and microorganism. Sugarcane wasobtained from Mandal Agricultural Office, Jangareddygudem,West Godavari Distract, Andhra Pradesh. S.cerevisiae cellgrowth was carried out at 30oC in yeast extract peptonedextrose (YPD) liquid nutrient medium containing 20 g/Lglucose, 10 g/L yeast extract, 20 g/L peptone. Sugarcane

juice containing a total sugar of 150 g/L was obtained bysqueezing of sugarcane in mechanical mini crusher. Molasseswas obtained from GMR distilleries, Sankili, SrikakulumDistrict, Andhra Pradesh. All media were autoclaved at121oC for 15 min.

Preparation of supports by sugarcane pieces for yeastimmobilization. First, sugarcane was defrosted afterstorage at −20oC. Sugarcane pieces were obtained byremoval of the external part of the hard skin and cuttinginto small pieces of 1 cm length. Delignification wasperformed according to Bardi and Koutinas [18] andKopsahelis et al. [19], by which the sugarcane pieceswere mixed with 1% NaOH solution for 4 hr to removethe lignin present inside the material. After that thedelignified pieces were washed well with water, and thensterilized at 121oC for 15 min. Cell immobilization onsugarcane pieces was carried out by suspending about4 × 109 yeast cells in 200 mL of YPD medium, and mixedwith 60 g of sterilized delignified sugarcane pieces. Themixture was allowed for culture about 16 hr. The fermentedliquid was then decanted, the immobilized biocatalyst waswashed twice with 200 mL of fresh YPD medium andsugarcane-supported biocatalyst was used for the followingrepeated batch fermentation [14].

Fermentation. An amount of 40 g (wet weight) of thesugarcane pieces containing yeast biocatalyst and 100 mLof sugarcane juice medium was added in a 500 mL flaskand anaerobic repeated batch fermentation was successivelycarried out at 30oC by adding fresh medium at each cycle.The fermented liquid was decanted at the end of eachfermentation batch, the biocatalyst was washed with100 mL of the sugarcane juice medium and then freshmedium was added for the next fermentation batch.Samples of the fermented liquids were collected andanalysed for the concentration of ethanol and residualsugar. Fermentations of diluted molasses were carried outin the same way at 30oC. All experiments were carried outin triplicate.

Analytical methods. The determinations were carriedout for cell viability using the methylene blue method[22] and total reducing sugar in acid hydrolysate (1.2 MHCl for 10 min at 60oC), using the 3,5-dinitrosalicylicacid method [23]. The sugar concentration was calculatedusing standard curves and expressed as gram sugar perlitre. For the biomass assays, cells were washed byvacuum filtration and dried 70oC until constant weight andexpressed as grams per litre in the final medium. Thesamples of fermented broth derived at various intervalsduring the period of experimentation were analyzed foralcohol levels using gas chromatography (GC-2014;Shimadzu, Kyoto, Japan) with a flame ionization detector.

Study of Sugarcane Pieces as Yeast Supports for Ethanol Production 37

The samples were filtered through 0.2 µm micro-filters forethanol concentration before injection (column: Rtx-5Crossbond 5% diphenyl/95% dimethyl poly siloxane, columntemperature 120oC, injector temperature 150oC, detectortemperature 200oC, flow rate of carrier gas 5 µL/min). Theethanol yield (Yps) was calculated as the actual ethanolproduced and expressed as g ethanol per g total sugarutilized (g/g). The volumetric ethanol productivity Qp (g/L/hr) was calculated by ethanol concentration produced P(g/L) divided by fermentation time giving the highestethanol concentration. Periodic samples were subjected toalcohol estimation after distilling the samples. Samples atevery 48 hr interval were centrifuged (4,000 rpm, 10 min)and 2 mg of pellets were dispersed in 1 mL of distilledwater and observed under microscope for cell viabilityusing standard procedure of methylene blue staining. Averagenumber of viable cells present in five microscopic fieldswas expressed as percentage of viability.

Scanning electron microscopy (SEM). For themicroscopic studies, samples were transferred to vials andfixed with glutaraldehyde in 0.05 M/L phosphate buffer(pH 7.2) for 24 hr at 4oC and post-fixed in 2.5× aqueousosmium tetra oxide in the above mentioned buffer for2 hr. After post-fixation, the samples were dehydrated inseries of graded alcohol and dried to critical point ofdrying (CPD, Model K850; Emitech, Ashford, England)with an Electron Microscopy Science CPD unit. Later, thedried samples were mounted over the stubs with double-sided conductivity tape. Finally, a thin layer of platinumwas passed through the sample using an automated sputtercoater (JFC-1600; Jeol, Tokyo, Japan) for about 90 sec.Then samples were scanned under SEM (JSM 6610LV;Jeol) at various magnifications using 5 kV (acceleratingvoltage) at Advanced Analytical Laboratory (NationalFacility), Andhra University, Visakhapatnam, India [7, 8, 15].

Results and Discussion

Yeast immobilization was shown by the scanning electronmicrographs. It was observed that high populations ofyeast cells homogenously adhered to the surface of sugarcanepieces even after 10 repeated batch fermentations. Figs.1~4 showing the proliferation of the yeast cells withinmatrix tissue structure. Immobilization did not affect theviability as immobilized yeast retained 90~95% of itsviability. Also our results are in agreement with others inthis regard that the application of low continuous stirringspeeds during the immobilization could lead to betterimmobilization performance [15, 24].

Based on these immobilization techniques, the sugarcanepieces are believed to immobilize yeast cells as a result ofnatural entrapment into the porous structure of sugarcanematerials and due to physical adsorption by electrostatic

Fig. 1. Scanning electron microscope image of inside theparenchyma of sugarcane pieces (A) control and (B)closer view of inside the parenchyma of sugarcanepieces (control).

Fig. 2. Scanning electron micrograph of the middle part of thesupport after yeast immobilization. Cross sectionalview showing absorbed yeast cells in parenchymacells at first cycle.

38 Babu et al.

forces between the cell membrane and the carrier. Also,these observations indicate that cell retention is due tothe action of capillary forces during the process ofimmobilization, which pull the cells to approach and keepclose contact with the surface and through the channelswhere they can be entrapped or attached, and multiply.The flow of cells over a porous support causes a pressuredifferential within the vessels causing the cells to be takeninto the vessels. High populations of immobilized yeastcells on the external surface of the support were observedafter ten batches of fermentation. This can be explainedby the greater nutrient availability (sugars) near thesurface of the support. In addition, the greater nutrientavailability (sugars) in the cavities of parenchyma cells isalso an attraction to yeast and enable yeasts tend tomigrate into the inner of parenchyma cells and budding inimmobilization process [14].

Repeated fermentation batch. Yeast cell immobilizationon the sugarcane and suitability of the immobilizedbiocatalysts for alcoholic fermentation was confirmed bysatisfactory operational stability during repeated batchfermentations of sugarcane juice. The variation of viablecell was measured by the number of colony-forming units(cfu) in the fermentation broth (cell leakage) and on thebiocatalyst (immobilized cells) during the repeated batchfermentation of sugarcane juice and molasses. Accordingto Fig. 5, enumeration of immobilized viable cells onsugarcane immediately after immobilization indicated thatthe microbial populations were 1010 cells/g of biocatalysts.At the end of the third fermentation batch using sugarcanejuice, the immobilized cells increased to 1011 cells/g ofbiocatalyst. It was observed that the number of cellsattached to the support increased during the period, whilefewer and fewer yeast cells leaked into fermentation broth.Therefore, the immobilization of the cells is believed to bea time depending process. This occurrence could be dueto two main factors: cell multiplication and the formationof a strong and irreversible adhesion.

According to the immobilization theory Tanaka andKawamoto [21], this time-dependence condition might bealso influenced by interactions both Van der Waals forcesand electrostatic forces. It was obvious that high densitiesof immobilized cells were achieved, which keep theirviability at stable levels during successive fermentationbatches, for comparison, fermentations of sugarcane juicewere carried out using free yeast cells and the viable cellsin the fermentation broth was only about 2~4 × 108 cells/mL, far lower than immobilized cells in our biocatalystsystem. Effective immobilization was also established bythe ability of the biocatalyst (after washing to remove freecells) to perform efficiently repeated batch fermentations

Fig. 3. Scanning electron microscope image of yeast (NCIM3640) inside the parenchyma of sugarcane pieces aftersixth cycle.

Fig. 5. Viable cell counts of free and immobilized cells duringrepeated batch fermentations of sugarcane juice (as×1010 colony-forming unit [cfu]/g wet sugarcane or×107 cfu/mL fermentation broth).

Fig. 4. Scanning electron microscope image of yeast (NCIM3640) inside the parenchyma of sugarcane pieces aftersixth cycle (closer view).

Study of Sugarcane Pieces as Yeast Supports for Ethanol Production 39

of sugarcane juice and molasses in the production of bioethanol.

The kinetic parameters obtained after repeated batchfermentations of sugarcane juice and molasses for tencycles using our immobilization system were shown inTable 1 (the data showed only 6 cycles). These resultsshowed that fermentation times for all the media investigatedwere lower than 34 hr and stable. Fermentation timeswere low even during the first batch, indicating that nosignificant period was needed for adaptation of thebiocatalyst in the fermentation environment. Ethanolconcentrations (about 73.25~76.28 g/L in average value),and ethanol productivities (about 2.24~2.36 g/L/hr inaverage value) were high and stable, and residual sugarconcentrations were low in all fermentations (0.90~3.25 g/L)with conversions ranging from 97.67 to 99.80%, showingefficiency (91.57~95.55%) and operational stability of thebiocatalyst for ethanol fermentation. However, as some ofthe carbon sources is used for biomass and volatile by-products generation, the actual ethanol yield is about90~95% (0.46 g ethanol/g sugar) of the theoretical one[25, 26]. In most of the distilleries in Mexico, the yield ofethanol from molasses ranged from 0.33 up to 0.38 gethanol/g reducing sugars. In the case of the fermentationof sugarcane juice (161.6 g of initial sugar/L) and molasses(151.6 g of initial sugar/L) by the biocatalyst that yeastimmobilized on sugarcane pieces, the actual ethanol yieldsof this work were 0.47 g/g and 0.48 g/g, respectively,which are considered as acceptable values. Comparedwith those obtained with free cells, similar concentrationsof ethanol were obtained, however, the main differenceobserved was the higher fermentation rate of immobilizedcells, and higher ethanol productivity and fermentation

efficiency obtained.It is known that yeast cells can also be entrapped in

calcium alginate and the resulting immobilized yeast canbe used for fast paced fermentations. Numerous literaturereferences describe this technique as it is applied usuallyin laboratory scale. Some efforts, however, have also beenmade to commercialize this technique. One of the bestknown is probably that of Kyowa Hakko in Japan [27],where growing cells of S. cerevisiae immobilized in calciumalginate gel beads were employed in fluidized bed reactorsfor continuous ethanol fermentation from cane molassesand other sugar sources. According to the report, theethanol productivity was more than 50 g ethanol/L gel/hrand prolonged life stability for more than one-half year.Cell concentration in the carrier was estimated over 250 gdry cell/L gel. As a result, it was confirmed that 8~10%(v/v) ethanol-containing broth was continuously producedfrom non-sterilized diluted cane molasses for over one-half year. The productivity of ethanol was calculated as0.6 L ethanol per L of reactor volume one day with a95% conversion yield versus the maximum theoreticalyield for the case of 8.5% (v/v) ethanol broth. The resultsof this work were generally comparable with these of theprevious studies in the fermentation rates, yields andefficiencies, and contributed to an improved quality of thedistillate due to the 10~11.5% (v/v) higher ethanol content[14].

On other hand, there are several weaknesses of alginateimmobilized yeast for commercial scale operations. Amajor difficulty with alginate entrapment is the manner inwhich the particles are formed. A process plant that utilizesalginate entrapped yeast must have specially designedequipment just to produce these beads. Furthermore, there

Table 1. Kinetic parameters of the anaerobic repeated batch fermentation of sugarcane juices and molasses with NCIM 3640immobilized on sugarcane pieces at 30oC

MediaNo. ofcycles

t(hr)

Initial sugar(g/L)

Residual sugar(g/L)

Conversion(%)

P(g/L)

Qp

(g/L/hr)Yps

(g/g)Ey

(%)

Sugar cane juice 1 33 160.2 2.25 98.59 75.25 2.28 0.46 91.922 32 160.6 2.85 98.22 75.15 2.34 0.46 91.573 34 161.2 1.10 99.31 75.54 2.22 0.46 91.704 34 160.6 0.90 99.43 75.60 2.22 0.46 92.125 34 161.2 1.78 99.14 76.20 2.24 0.47 92.506 34 161.6 1.00 99.38 76.28 2.24 0.47 92.37

Molasses 1 31 150.2 3.25 97.83 73.25 2.36 0.48 95.432 32 150.6 1.85 98.77 73.51 2.29 0.48 95.523 32 151.2 2.10 98.61 72.65 2.27 0.48 94.004 31 150.6 2.50 98.33 73.00 2.35 0.48 94.855 32 151.2 2.68 98.22 73.36 2.29 0.48 94.946 32 151.6 2.98 98.03 73.62 2.30 0.48 95.03

Sugar cane juice a 48 160.6 3.25 97.97 75.14 1.56 0.46 91.55Molasses b 48 150.2 4.20 97.20 72.12 1.50 0.48 93.96

t, fermentation time; P, ethanol concentration; Qp, volumetric ethanol productivity; Yps, ethanol yield; Ey (%), fermentation efficiency.aSugarcane juice control (free cells).bMolasses control (free cells).

40 Babu et al.

is a potential risk for contaminating the yeast with wildmicroorganisms.

A second major difficulty for alginate beads used on acommercial scale is in the physical strength of the beads.The beads are soft and easily compressible. Operatinglarge fermentation columns can be a problem and fastdown flow process streams are difficult to handle. A thirddifficulty with entrapment is the diffusion limitations whichslowdown the accessibility of the substrate in contact withthe yeast inside the bead. Finally, if the system becomescontaminated or otherwise disturbed so that a continuousoperation must be discontinued, the whole lot of columnmaterial (alginate together with the yeast) must be discarded.No reuse is possible. However, the spent sugarcaneimmobilized supports can be used as protein enriched(SCP production) animal feed for reusing. Therefore, incomparison with Ca-alginate entrapped yeast, this sugarcaneimmobilized biocatalysts is believed to be competitive foran industrial process [14]. The biocatalysts prepared bynatural materials for the production of alcohol have alsobeen extensively studied, such as apple pieces [7], orangepeel [11], dried figs [28] and spent grains [19]. Most ofthe fermentation batches resulted in glucose consumptionof 98.00~99.00% for the juice containing 134.00~187.00 gof sugar/L, and stable ethanol production with ethanolproductivity ranging from 26.00 to 110.40 g/L/day at 30oC.Anyway, the results presented in this study, according toinitial concentration of sugars in the must, showed that thesugarcane supported biocatalyst was equally efficient tothat described in the literature for ethanol fermentation[14].

The results of this study mainly demonstrated the potentialapplications of the sugarcane as a matrix material foryeast immobilization and provided a good investigation into the possibilities of immobilized yeast cells in ethanolproduction. Sugarcane pieces were found suitable assupport for yeast cell immobilization in ethanol industry.The sugarcane immobilized biocatalysts showed highfermentation activity. Although ethanol fermentations havebeen only carried out on sterilized sugarcane juice, thefermentation technologies presented above could be appliedin fresh juice as well. The immobilized yeast would dominatein the fermentation broth due to its high populations andlower fermentation time, therefore, the development ofother wild yeast and bacteria that may exist in the freshbroth would be inhibited [8]. Owing to the low price ofthe support material and its abundance in nature, reuseavailability make this biocatalyst attractive in the ethanolproduction as well as in wine making and beer production.Also, the particles form of the support give the possibilityfor fermentations using feed batch bioreactors and separationof biocatalyst employing centrifuge separators or separationafter removal of the supernatant liquid. From this study,we can conclude that the sugar cane pieces are well

suitable for yeast immobilization and can be serve as agood biocatalyst for ethanol fermentation. This method isvery economical and easy method for ethanol productionand suitable for ethanol industry. The sugarcane immobilizedbiocatalyst showed high fermentation activity. These resultsshowed that fermentation time required for all the mediainvestigated were lower than 35 hr and stable. Fermentationtimes were low even during the first batch, indicating thatno significant period was needed for adaptation of thebiocatalyst in the fermentation environment. Ethanolconcentrations (about 76.28~73.25 g/L in average value),and ethanol productivities (about 2.24~2.36 g/L/hr inaverage value) were high and stable, and residual sugarconcentrations were low in all fermentations (0.90~3.25 g/L)with conversions ranging from 97.67 to 99.80%, showingefficiency (91.57~95.55%) and operational stability of thebiocatalyst for ethanol fermentation. The immobilized yeastswere dominating in the fermentation broth due to its highpopulation and hence lower fermentation time. Thedevelopment of other wild yeast and bacteria that mayexist in the fresh broth was effectively inhibited. The lowprice of the support and its abundance in nature, reuseavailability make this biocatalyst attractive in the ethanolproduction. Finally, this method is very economical, easyand rate of fermentation has also shortened when comparedwith other popular cell immobilization protocols.

Acknowledgements

One of the authors (Mr. N. Kishore Babu) is thankful toRajiv Gandhi National Fellowship, New Delhi for providingresearch fellowship during this period.

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